As used herein, the term "blood products" includes whole blood and cellular components derived from blood, including erythrocytes (red blood cells) and platelets.
There are more than thirty blood group (or type) systems, one of the most important of which is the ABO system. This system is based on the presence or absence of antigens A and/or B. These antigens are found on the surface of erythrocytes and on the surface of all endothelial and most epithelial cells as well. The major blood product used for transfusion is erythrocytes, which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen. Blood of group A contains antigen A on its erythrocytes. Similarly, blood of group B contains antigen B on its erythrocytes. Blood of group AB contains both antigens, and blood of group O contains neither antigen.
The blood group structures are glycoproteins or glycolipids and considerable work has been done to identify the specific structures making up the A and B determinants or antigens. It has been found that the blood group specificity is determined by the nature and linkage of monosaccharides at the ends of the carbohydrate chains. The carbohydrate chains are attached to a peptide or lipid backbone which is embedded in the lipid bi-layer of the membrane of the cells. The most important (immuno-dominant or immuno-determinant) sugar has been found to be N-acetylgalactosamine for the type A antigen and galactose for the type B antigen.
There are three recognized major sub-types of blood type A. These sub-types are known as A.sub.1, A intermediate (A.sub.int) and A.sub.2. There are both quantitative and qualitative differences which distinguish these three sub-types. Quantitatively, A.sub.1 erythrocytes have more antigenic A sites, i.e., terminal N-acetylgalactosamine residues, than A.sub.int erythrocytes which in turn have more antigenic A sites than A.sub.2 erythrocytes. Qualitatively, the transferase enzymes responsible for the formation of A antigens differ biochemically from each other in A.sub.1, A and A.sub.2 individuals. Some A antigens found in A.sub.1 cells contain dual A antigenic sites.
Blood of group A contains antibodies to antigen B. Conversely, blood of group B contains antibodies to antigen A. Blood of group AB has neither antibody, and blood group O has both. A person whose blood contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s). If a person receives a transfusion of blood of an incompatible group, the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis (the destruction of red blood cells) may result therefrom.
In order to avoid red blood cell agglutination, transfusion reactions and hemolysis, transfusion blood type is cross-matched against the blood type of the transfusion recipient. For example, a blood type A recipient can be safely transfused with type A blood which contains compatible antigens. Because type O blood contains no A or B antigens, it can be transfused into any recipient with any blood type, i.e., recipients with blood types A, B, AB or O. Thus, type O blood is considered "universal", and may be used for all transfusions. Hence, it is desirable for blood banks to maintain large quantities of type O blood. However, there is a paucity of blood type O donors. Therefore, it is useful to convert types A, B and AB blood to type O blood in order to maintain large quantities of universal blood products.
In an attempt to increase the supply of type O blood, methods have been developed for converting certain type A, B and AB blood to type O blood. For example, U.S. Pat. No. 4,609,627 entitled "Enzymatic Conversion of Certain Sub-Type A and AB Erythrocytes" ("the '627 Patent"), which is incorporated herein by reference, is directed to a process for converting A.sub.int and A.sub.2 (including A.sub.2 B erythrocytes) to erythrocytes of the H antigen type, as well as to compositions of type B erythrocytes which lack A antigens, which compositions, prior to treatment, contained both A and B antigens on the surface of said erythrocytes. The process for converting A.sub.int and A.sub.2 erythrocytes to erythrocytes of the H antigen type which is described in the '627 Patent includes the steps of equilibrating certain sub-type A or AB erythrocytes, contacting the equilibrated erythrocytes with purified chicken liver .alpha.-N-acetylgalactosaminidase enzyme for a period sufficient to convert the A antigen to the H antigen, removing the enzyme from the erythrocytes and re-equilibrating the erythrocytes. As described in the '627 Patent, .alpha.-N-acetylgalactosaminidase obtained from an avian liver (specifically, chicken liver) source was found to have superior activity in respect of enzymatic conversion or cleavage of A antigenic sites.
Prior to the present invention, it was necessary to purify the enzyme from an avian liver source, a process which is time consuming and can be expensive. Hence, a need has arisen to develop an enzyme source which is more readily available. In addition, a need has arisen to develop an enzyme useful in blood product conversion which enzyme is cost-efficient.
A simplified purification process is described in a related application, Ser. No. 07/964,756, filed Oct. 22, 1992, entitled "Preparation of Enzyme for Conversion of Sub-Type A and AB Erythrocytes". This process, as described in the related application, utilizes chicken liver as a source of enzyme and, therefore, requires a number of purification steps. Despite this simplified process, it is still desirable to provide a more readily available and controlled source of enzyme, that being cloned and expressed enzyme. This would provide an enzyme source which is more consistent and which is readily purified at less cost and expense, with a still further reduced number of purification steps. Additionally, a recombinant, cloned enzyme allows for specific protein sequence modifications, which can be introduced to generate an enzyme with optimized specific activity, substrate specificity and pH range.
.alpha.-N-acetylgalactosaminidase enzymes are characterized (and thereby named) by their ability to cleave N-acetylgalactosamine sugar groups. In isolating or identifying these enzymes, their activity is assessed in the laboratory by evaluating cleavage of synthetic substrates which mimic the sugar groups cleaved by the enzymes, with p-nitrophenylglycopyranoside derivatives of the target sugar groups being commonly used. Although very useful in enzyme identification and isolation procedures (the quantitative cleavage of these synthetic substrates can be used to readily distinguish (and thereby identify) enzymes isolated from different sources), these synthetic substrates are simple structurally and small-sized and mimic only a portion of the natural glycoproteins and glycolipid structures which are of primary concern, those being the A antigens on the surface of cells.
A natural glycolipid substrate, originally isolated from sheep erythrocytes, is the Forsmann antigen (globopentaglycosylceramide). The Forsmann antigen substrate appropriately mimics the natural A antigen glycolipid structures and is therefore utilized to predict the activity of .alpha.-N-acetylgalactosaminidase enzymes against the A antigen substrate. Isolated Forsmann antigen glycolipids have been shown to inhibit hemolysis of sheep red cells by immune rabbit anti-A serum in the presence of serum complement.
.alpha.-N-acetylgalactosaminindase enzyme has been isolated from a number of sources besides chicken liver (described above), including bacteria, mollusks, earthworms, and human liver. The human .alpha.-N-acetylgalactosaminidase enzyme has been purified, sequenced, cloned and expressed. For example, in "Human .alpha.-N-Acetylgalactosaminidase--Molecular Cloning, Nucleotide Sequence and Expression of a Full-length cDNA", by Wang et al., in The Journal of Biological Chemistry, Vol. 265, No. 35, pages 21859-21866 (Dec. 15, 1990), the cDNA encoding human .alpha.-N-acetylgalactosaminidase was sequenced. In addition, in "Molecular Cloning of a Full-Length cDNA for Human .alpha.-N-Acetylgalactosaminidase (.alpha.-Galactosidase B)", by Tsuji et al., in Biochemical And Biophysical Research Communications, Vol. 163, No. 3, pages 1498-1504 (Sep. 29, 1989), the cDNA encoding human .alpha.-N-acetylgalactosaminidase was sequenced. Both the nucleotide sequence and the amino acid sequence of human .alpha.-N-acetylgalactosaminidase is published therein. Further, PCT Application No. WO 92/07936 discloses the cloning and expression of the cDNA which encodes human .alpha.-N-acetylgalactosaminidase.
Although human .alpha.-N-acetylgalactosaminidase has been purified, sequenced, cloned and expressed, it is not appropriate for use in removing A antigens from the surface of cells in blood products. In determining whether an enzyme is appropriate for use in removing A antigens from the surface of cells, one must consider the following enzyme characteristics, particularly with respect to the Forsmann antigen substrate: substrate specificity, specific activity or velocity of the substrate cleavage reaction, and pH optimum. Substrate specificity is measured in the Km value, which measures the binding constant or affinity of an enzyme for a particular substrate. The lower a Km value, the more tightly an enzyme binds its substrate. The velocity of an enzyme cleavage reaction is measured in the Vmax, the reaction rate at a saturating concentration of substrate. A higher Vmax indicates a faster cleavage rate. The ratio of these two parameters, Vmax/Km, is a measure of the overall efficiency of an enzyme in reacting with (cleaving) a given substrate. A higher Vmax/Km indicates greater enzyme efficiency. For successful and clinically applicable removal of A antigens from the surface of cells, the enzyme must be sufficiently active at or above a pH at which the cells being treated can be maintained. The procedure described in the '627 patent calls for treatment of cells at or above a pH of 5.6. Therefore, the pH optimum of an appropriate enzyme must still provide reasonable enzyme activity at this pH. These specific characteristics (Vmax/Km, Vmax, Km and pH optimum) are reported for the human .alpha.-N-acetylgalactosaminidase enzyme in "Studies on Human Liver .alpha.-galactosidases", by Dean et al. in The Journal of Biological Chemistry, Vol. 254, No. 20, pages 10001-10005 (1979).
The Vmax/Km value for the Forsmann antigen of human .alpha.-N-acetylgalactosaminidase is 0.46, as compared to a Vmax/Km value of 5.0 for the chicken liver enzyme, indicating an approximately ten-fold difference in efficiency. The Km is lower and the Vmax is higher for the chicken liver enzyme, compared to the human enzyme. Further, human .alpha.-N-acetylgalactosaminidase has a pH optimum for the Forsmann antigen of 3.9, compared to 4.7 for chicken liver .alpha.-N-acetylgalactosaminidase. By all of these enzyme characteristics, human .alpha.-N-acetylgalactosaminidase enzyme is not suitable for removal of A antigens, particularly when compared to the chicken liver enzyme.
As a result, a need still existed to develop an enzyme which is capable of removing A antigens from the surface of cells in blood products, wherein said enzyme is readily available and cost-efficient.
It is therefore an object of this invention to provide a recombinant enzyme for use in the removal of A antigens from the surface of cells in blood products.
It is another object of this invention to provide a recombinant enzyme for use in the removal of A antigens from the surface of cells in blood products wherein said enzyme is readily available and may be manufactured on a cost-efficient basis.
It is a further object of this invention to provide methods of cloning and expressing a recombinant enzyme useful in the removal of A antigens from the surface of cells in blood products.
It is yet another object of this invention to provide a method of removing A antigens from the surface of cells in blood products using a recombinant enzyme.